Activation of CpxRA in Haemophilus ducreyi Primarily Inhibits the Expression of Its Targets, Including Major Virulence Determinants

Gangaiah, Dharanesh; Zhang, Xinjun; Fortney, Kate R.; Baker, Beth; Liu, Yunlong; Munson, Robert S.; Spinola, Stanley M.

doi:10.1128/jb.00372-13

Cited by 33 publications

(69 citation statements)

References 53 publications

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“…For example, in Haemophilus ducreyi, the Cpx response appears not to be as tightly linked to protein misfolding but rather was shown to control the expression of many genes, including several major virulence determinants, predominantly in a negative fashion (22,23). Similarly, for Vibrio cholerae El Tor N16961, recent evidence indicates that the Cpx pathway may be involved in sensing and mediating adaptation to high salinity in addition to responding to misfolded envelope proteins that contain aberrant disulfide bonds (24).…”

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confidence: 99%

The Vibrio cholerae Cpx Envelope Stress Response Senses and Mediates Adaptation to Low Iron

2014

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The Cpx pathway, a two-component system that employs the sensor histidine kinase CpxA and the response regulator CpxR, regulates crucial envelope stress responses across bacterial species and affects antibiotic resistance. To characterize the CpxR regulon in Vibrio cholerae, the transcriptional profile of the pandemic V. cholerae El Tor C6706 strain was examined upon overexpression of cpxR. Our data show that the Cpx regulon of V. cholerae is enriched in genes encoding membrane-localized and transport proteins, including a large number of genes known or predicted to be iron regulated. Activation of the Cpx pathway further led to the expression of TolC, the major outer membrane pore, and of components of two RND efflux systems in V. cholerae. We show that iron chelation, toxic compounds, or deletion of specific RND efflux components leads to Cpx pathway activation. Furthermore, mutations that eliminate the Cpx response or members of its regulon result in growth phenotypes in the presence of these inducers that, together with Cpx pathway activation, are partially suppressed by iron. Cumulatively, our results suggest that a major function of the Cpx response in V. cholerae is to mediate adaptation to envelope perturbations caused by toxic compounds and the depletion of iron. Perturbations of the bacterial cell envelope can induce cell envelope stress responses. During this process, signal transduction pathways, such as two-component systems (TCS), are necessary for transducing the information from the cell envelope to the cytoplasm for gene expression regulation (1). TCS are the most prevalent signaling pathways in bacteria, and they are involved in the responses to different inducing cues, mainly related to stress responses and environmental changes (2). The Cpx envelope stress response is an example of a TCS and is composed of the sensor histidine kinase CpxA and the response regulator CpxR (3). This system senses envelope stress and regulates the expression of genes involved in maintaining cell envelope homeostasis to increase bacterial survival under adverse conditions (4).CpxA is an inner membrane (IM) protein that autophosphorylates upon detecting an inducing cue and then becomes a phosphodonor and transfers a phosphoryl group to a conserved aspartate of its response regulator, CpxR (3, 5). CpxR phosphorylation leads to the alteration in transcription of multiple genes by the direct binding of CpxR to DNA (5, 6). In addition, CpxR phosphorylation is modulated indirectly by a periplasmic protein, CpxP, which reduces CpxA activity (6, 7) through a possible direct interaction with the periplasmic sensing domain of CpxA (8-11).The Cpx system in Escherichia coli senses misfolded proteins in the bacterial cell envelope and regulates protein folding and degrading factors, such as DegP, DsbA, and PpiD, involved in the alleviation of the envelope stress (12-15). For example, some of the inducing cues of this pathway are aggregated uropathogenic E. coli P pilus subunits and subunits of the enteropathogenic E. coli bund...

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The Vibrio cholerae Cpx Envelope Stress Response Senses and Mediates Adaptation to Low Iron

2014

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“…The amplification efficiency was determined for each primer pair; all primer pairs had greater than 95% efficiency. The expression levels of target genes were normalized to that of dnaE using primers described previously (11). The fold change in expression was calculated as (E target )…”

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confidence: 99%

“…qRT-PCR was performed to amplify internal gene-specific fragments ranging from 70 to 200 bp of HD0518, rluA, dsbA, degP, and HD0192 using primers P13/ P14, P15/P16, P17/P18, P19/P20, and P21/P22, respectively (see Table S1 in the supplemental material); qRT-PCR analysis of HD0430, HD0930, hfq, and ompP2B was performed using primers described previously (11). The amplification efficiency was determined for each primer pair; all primer pairs had greater than 95% efficiency.…”

Section: Methodsmentioning

confidence: 99%

“…For induction of FLAG-tagged RpoE expression, 35000HP(pDG11) and 35000HP(pDG10) were grown to mid-log phase (OD 660 ϭ 0.2) and then treated with 200 ng/ml of ATc, harvested, and analyzed as described above. Western blotting was performed on whole-cell lysates of 35000HP-(pDG11) and 35000HP(pDG10) before and 5, 10, and 20 min after adding ATc, using the monoclonal anti-FLAG M2 antibody (Sigma) (11).…”

Section: Methodsmentioning

confidence: 99%

“…The H. ducreyi genome encodes homologues of CpxRA, which is the only obvious intact TCS encoded in the genome. Activation of CpxR by deletion of cpxA downregulates the majority of its targets, including multiple virulence determinants, and attenuates the virulence of H. ducreyi in humans (10)(11)(12). However, deletion of cpxR does not affect the expression of virulence determinants or reduce the virulence of the organism in humans, suggesting that CpxRA is dispensable for H. ducreyi infection (13).…”

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Haemophilus ducreyi RpoE and CpxRA Appear To Play Distinct yet Complementary Roles in Regulation of Envelope-Related Functions

et al. 2014

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Haemophilus ducreyi causes the sexually transmitted disease chancroid and a chronic limb ulceration syndrome in children. In humans, H. ducreyi is found in an abscess and overcomes a hostile environment to establish infection. To sense and respond to membrane stress, bacteria utilize two-component systems (TCSs) and extracytoplasmic function (ECF) sigma factors. We previously showed that activation of CpxRA, the only intact TCS in H. ducreyi, does not regulate homologues of envelope protein folding factors but does downregulate genes encoding envelope-localized proteins, including many virulence determinants. H. ducreyi also harbors a homologue of RpoE, which is the only ECF sigma factor in the organism. To potentially understand how H. ducreyi responds to membrane stress, here we defined RpoE-dependent genes using transcriptome sequencing (RNASeq). We identified 180 RpoE-dependent genes, of which 98% were upregulated; a major set of these genes encodes homologues of envelope maintenance and repair factors. We also identified and validated a putative RpoE promoter consensus sequence, which was enriched in the majority of RpoE-dependent targets. Comparison of RpoE-dependent genes to those controlled by CpxR showed that each transcription factor regulated a distinct set of genes. Given that RpoE activated a large number of genes encoding envelope maintenance and repair factors and that CpxRA represses genes encoding envelope-localized proteins, these data suggest that RpoE and CpxRA appear to play distinct yet complementary roles in regulating envelope homeostasis in H. ducreyi.

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The Cpx Inner Membrane Stress Response

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Raivio

2016

Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria

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Activation of CpxRA in Haemophilus ducreyi Primarily Inhibits the Expression of Its Targets, Including Major Virulence Determinants

Cited by 33 publications

References 53 publications

The Vibrio cholerae Cpx Envelope Stress Response Senses and Mediates Adaptation to Low Iron

The Vibrio cholerae Cpx Envelope Stress Response Senses and Mediates Adaptation to Low Iron

Haemophilus ducreyi RpoE and CpxRA Appear To Play Distinct yet Complementary Roles in Regulation of Envelope-Related Functions

The Cpx Inner Membrane Stress Response

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